Antisense modulation of ara70 expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of ARA70. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding ARA70. Methods of using these compounds for modulation of ARA70 expression and for treatment of diseases associated with expression of ARA70 are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods formodulating the expression of ARA70. In particular, this inventionrelates to antisense compounds, particularly oligonucleotides,specifically hybridizable-with nucleic acids encoding ARA70. Sucholigonucleotides have been shown to modulate the expression of ARA70.

BACKGROUND OF THE INVENTION

[0002] Steroid, thyroid and retinoid hormones produce a diverse array ofphysiologic effects through the regulation of gene expression. Uponentering the cell, these hormones bind to a unique-group ofintracellular nuclear receptors which have been characterized asligand-dependent transcription factors. This complex then moves into thenucleus where the receptor and its cognate ligand interact with thetranscription pre-initiation complex affecting its stability andultimately the rate of transcription of the target genes. Theinteractions of the liganded receptor with the specific elements in thepromoter region are mediated by two classes of molecules; corepressors,which inhibit transactivation and coactivators, which enhancetransactivation (Shibata et al., Recent Prog. Horm. Res., 1997, 52,141-164).

[0003] ARA70 (also known as androgen receptor (AR) activator, ELE1,RET/PCT3, RFG for RET-fused gene and NCOA4 for nuclear receptorcoactivator 4) is a member of the growing family of transcriptionalcoactivators. ARA70 was first isolated as a coactivator for the androgenreceptor (AR) in human prostate cancer cells and has since been shown toact as a ligand-enhanced coactivator for the peroxisomeproliferator-activated receptor gamma, another nuclear hormone receptor,in these same cells (Heinlein et al., J. Biol. Chem., 1999, 274,16147-16152; Yeh and Chang, Proc. Natl. Acad. Sci. U.S.A., 1996, 93,5517-5521). ARA70 shows a broad tissue distribution of expression,including adipose tissue (Yeh and Chang, Proc. Natl. Acad. Sci. U.S.A.,1996, 93, 5517-5521) and the DNA and protein sequence are disclosed inthe PCT publication WO 97/44490 (Chang and Yeh, 1997).

[0004] Studies of androgen receptor regulation in prostate cancer celllines have revealed a pivotal role for ARA70 in sex hormone signaling.By properly interacting with the androgen receptor, it has beensuggested that ARA70 confers specificity of action between androgen andestrogen in the prostate (Miyamoto et al., Proc. Natl. Acad. Sci.U.S.A., 1998, 95, 11083-11088; Miyamoto et al., Proc. Natl. Acad. Sci.U.S.A., 1998, 95, 7379-7384; Yeh et al., Proc. Natl. Acad. Sci. U.S.A. ,1998, 95, 5527-5532).

[0005] Furthermore, cotransfection of ARA70 with the retinoblastomaprotein (Rb), a tumor suppressor, in the prostate cancer cell line DU145 has been shown to additively induce androgen receptortranscriptional activity by 13-fold with a concomitant reduction of cellgrowth (Yeh et al., Biochem. Biophys. Res. Commun., 1998, 248, 361-367).

[0006] ARA70 has also been identified in thyroid cancer cells, wherechromosomal rearrangement resulted in the fusion of the genomic regionof the RET tyrosine kinase to the 5′ terminal region of the ELE1 gene.The resulting protein was designated in this context as RET/PTC3 orRET-fused gene (Bongarzone et al., Genomics, 1997, 42, 252-259;Minoletti et al., Genes Chromosomes Cancer, 1994, 11, 51-57; Santoro etal., Oncogene, 1994, 9, 509-516). More recently, it has been discoveredthat there are two isoforms of the ELE1 gene to which the RET tyrosinekinase can be fused to produce ARA70. The expression of these isoforms,designated alpha and beta (beta being a deletion variant most likelycaused by the removal of one or more exons), thereby produce twoisoforms of the ARA70 coactivator (Alen et al., Mol. Endocrinol., 1999,13, 117-128).

[0007] Given the suggested role and expression pattern of ARA70 inprostate and thyroid carcinomas, the pharmacological modulation of ARA70activity and/or expression may be an appropriate point of therapeuticintervention in pathological conditions.

[0008] Currently, there are no known therapeutic agents whicheffectively inhibit the synthesis of ARA70. Consequently, there remainsa long felt need for agents capable of effectively inhibiting ARA70function.

[0009] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of ARA70 expression. Thetransfection of antisense ARA.sub.70 in order to block androgen receptortranscriptional activity is generally disclosed in U.S. Pat. No.5,789,170.

[0010] The present invention provides compositions and methods formodulating ARA70 expression, including modulation of the expression ofthe extended mRNA form of ARA70, known as RET-fused gene (RFG).

SUMMARY OF THE INVENTION

[0011] The present invention is directed to antisense compounds,particularly oligonucleotides, which are targeted to a nucleic acidencoding ARA70, and which modulate the expression of ARA70.Pharmaceutical and other compositions comprising the antisense compoundsof the invention are also provided. Further provided are methods ofmodulating the expression of ARA70 in cells or tissues comprisingcontacting said cells or tissues with one or more of the antisensecompounds or compositions of the invention. Further provided are methodsof treating an animal, particularly a human, suspected of having orbeing prone to a disease or condition associated with expression ofARA70 by administering a therapeutically or prophylactically effectiveamount of one or more of the antisense compounds or compositions of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention employs oligomeric antisense compounds,particularly oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding ARA70, ultimately modulating the amountof ARA70 produced. This is accomplished by providing antisense compoundswhich specifically hybridize with one or more nucleic acids encodingARA70. As used herein, the terms “target nucleic acid” and “nucleic acidencoding ARA70” encompass DNA encoding ARA70, RNA (including pre-mRNAand mRNA) transcribed from such DNA, and also cDNA derived from suchRNA. The specific hybridization of an oligomeric compound with itstarget nucleic acid interferes with the normal function of the nucleicacid. This modulation of function of a target nucleic acid by compoundswhich specifically hybridize to it is generally referred to as“antisense”. The functions of DNA to be interfered with includereplication and transcription. The functions of RNA to be interferedwith include all vital functions such as, for example, translocation ofthe RNA to the site of protein translation, translation of protein fromthe RNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity which may be engaged in or facilitated by the RNA.The overall effect of such interference with target nucleic acidfunction is modulation of the expression of ARA70. In the context of thepresent invention, “modulation” means either an increase (stimulation)or a decrease (inhibition) in the expression of a gene. In the contextof the present invention, inhibition is the preferred form of modulationof gene expression and mRNA is a preferred target.

[0013] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding ARA70. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding ARA70, regardless of the sequence(s) of such codons.

[0014] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0015] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0016] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. It has also beenfound that introns can also be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0017] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0018] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

[0019] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0020] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotides have been safely and effectively administered to humansand numerous clinical trials are presently underway. It is thusestablished that oligonucleotides can be useful therapeutic modalitiesthat can be configured to be useful in treatment regimes for treatmentof cells, tissues and animals, especially humans. In the context of thisinvention, the term “oligonucleotide” refers to an oligomer or polymerof ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimeticsthereof. This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0021] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about30 nucleobases (i.e. from about 8 to about 30 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides, even more preferably those comprising from about 12 toabout 25 nucleobases. As is known in the art, a nucleoside is abase-sugar combination. The base portion of the nucleoside is normally aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides are nucleosidesthat further include a phosphate group covalently linked to the sugarportion of the nucleoside. For those nucleosides that include apentofuranosyl sugar, the phosphate group can be linked to either the2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides,the phosphate groups covalently link adjacent nucleosides to one anotherto form a linear polymeric compound. In turn the respective ends of thislinear polymeric structure can be further joined to form a circularstructure, however, open linear structures are generally preferred.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

[0022] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0023] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chi-ral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thiono-alkylphosphonates,thionoalkylphosphotriesters, and borano-phosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acidforms are also included.

[0024] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; and 5,625,050, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

[0025] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside-linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

[0026] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

[0027] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0028] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃) —N(CH₃) —CH₂— and —O—N(CH₃) —CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0029] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br,CN, CF₃. OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylamino-ethoxyethoxy (also known in the art as2′-O-dimethylamino-ethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

[0030] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference in its entirety.

[0031] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine-bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in The Concise Encyclopedia Of PolymerScience And Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandteChemie, International Edition, 1991, 30, 613, and those disclosed bySanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain ofthese nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

[0032] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; and 5,681,941, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference, and U.S. Pat. No. 5,750,692, which iscommonly owned with the instant application and also herein incorporatedby reference.

[0033] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid(Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol orundecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118;Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al.,Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

[0034] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0035] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0036] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0037] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0038] The antisense compounds of the invention are synthesized in vitroand do not include antisense compositions of biological origin, orgenetic vector constructs designed to direct the in vivo synthesis ofantisense molecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0039] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0040] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 to Imbach et al.

[0041] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0042] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfoic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0043] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0044] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of ARA70 is treated by administering antisense compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

[0045] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding ARA70, enabling sandwich and other assays to easily beconstructed to exploit this fact. Hybridization of the antisenseoligonucleotides of the invention with a nucleic acid encoding ARA70 canbe detected by means known in the art. Such means may includeconjugation of an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection means. Kits using suchdetection means for detecting the level of ARA70 in a sample may also beprepared.

[0046] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0047] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful.

[0048] Compositions and formulations for oral administration includepowders or granules, suspensions -or solutions in water or non-aqueousmedia, capsules, sachets or tablets. Thickeners, flavoring agents,diluents, emulsifiers, dispersing aids or binders may be desirable.

[0049] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0050] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0051] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0052] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0053] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0054] Emulsions

[0055] The compositions of the present invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μmin diameter. (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

[0056] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0057] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0058] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0059] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0060] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0061] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0062] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of reasons of ease of formulation,efficacy from an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0063] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington Is Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0064] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0065] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0066] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0067] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories - surfactants, fatty acids,bile salts, chelating agents, and non-chelating non-surfactants (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92)Each of these classes has been discussed above.

[0068] Liposomes

[0069] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0070] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. -Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0071] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0072] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0073] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranes.As the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0074] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration, liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0075] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0076] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0077] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0078] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0079] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0080] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

[0081] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765).

[0082] Various liposomes comprising one or more glycolipids are known inthe art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0083] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

[0084] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0085] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0086] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

[0087] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0088] If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0089] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0090] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0091] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0092] Penetration Enhancers

[0093] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0094] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0095] Surfactants: In connection with the present invention,surfactants (or “surface-active agents”) are chemical entities which,when-dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. In addition to bile salts and fattyacids, these penetration enhancers include, for example, sodium laurylsulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetylether) (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p.92); and perfluorochemical emulsions, such as FC-43.Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0096] Fatty acids: Various fatty acids and their derivatives which actas penetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0097] Bile salts: The physiological role of bile includes thefacilitation of dispersion and absorption of lipids and fat-solublevitamins (Brunton, Chapter 38 in: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, NewYork, 1996, pp. 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus the term “bilesalts” includes any of the naturally occurring components of bile aswell as any of their synthetic derivatives. The bile salts of theinvention include, for example, cholic acid (or its pharmaceuticallyacceptable sodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783;.Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0098] Chelating Agents: Chelating agents, as used in connection withthe present invention, can be defined as compounds that remove metallicions from solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0099] Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

[0100] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

[0101] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0102] Carriers

[0103] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0104] Excipients

[0105] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

[0106] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0107] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0108] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0109] Other Components

[0110] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0111] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0112] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include, but are notlimited to, anticancer drugs such as daunorubicin, dactinomycin,doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil,melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine(CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages, 1206-1228). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. See, generally, The Merck Manual of Diagnosis and Therapy,15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and46-49, respectively). Other non-antisense chemotherapeutic agents arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

[0113] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0114] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0115] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

[0116] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxyand 2′-alkoxy Amidites

[0117] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, the standard cycle for unmodifiedoligonucleotides was utilized, except the wait step after pulse deliveryof tetrazole and base was increased to 360 seconds.

[0118] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

[0119] 2′-Fluoro Amidites

[0120] 2′-Fluorodeoxyadenosine Amidites

[0121] 2′-fluoro oligonucleotides were synthesized as describedpreviously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] andU.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, theprotected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine wassynthesized utilizing commercially available9-beta-D-arabinofuranosyladenine as starting material and by modifyingliterature procedures whereby the 2′-alpha-fluoro atom is introduced bya S_(N)2-displacement of a 2′-beta-trityl group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies and standard methods were used to obtain the5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0122] 2′-Fluorodeoxyguanosine

[0123] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0124] 2′-Fluorouridine

[0125] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

[0126] 2′-Fluorodeoxycytidine

[0127] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0128] 2′-O-(2-Methoxyethyl) Modified Amidites

[0129] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

[0130] 2,2′-Anhydro1-(beta-D-arabinofuranosyl)-5-methyluridine]

[0131] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60° C. at 1mm Hg for 24 h) to give a solid that was crushed to a light tan powder(57 g, 85% crude yield). The NMR spectrum was consistent with thestructure, contaminated with phenol as its sodium salt (ca. 5%). Thematerial was used as is for further reactions (or it can be purifiedfurther by column chromatography using a gradient of methanol in ethylacetate (10-25%) to give a white solid, mp 222-4° C.).

[0132] 2′-O-Methoxyethyl-5-methyluridine

[0133] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160° C. After heating for 48 hours at 155-160°C., the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product. Additional material was obtained byreworking impure fractions.

[0134] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0135] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

[0136]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0137] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by TLC by first quenching the TLC sample with the additionof MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

[0138]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0139] A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

[0140] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0141] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

[0142]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0143] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, TLC showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 ml), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

[0144]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0145]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (TLC showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

[0146] 2′-O-(Aminooxyethyl) Nucleoside Amidites and2′-O-(dimethylaminooxyethyl) Nucleoside Amidites

[0147] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

[0148] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

[0149] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0150] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.The solution was concentrated under reduced pressure to a thick oil.This was partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer was dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution was cooled to −10° C. The resultingcrystalline product was collected by filtration, washed with ethyl ether(3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of whitesolid. TLC and NMR were-consistent with pure product.

[0151]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0152] In a 2 L stainless steel, unstirred pressure reactor was addedborane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood andwith manual stirring, ethylene glycol (350 mL, excess) was addedcautiously at first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

[0153] 2′-O- ([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0154]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete,the.reaction was stirred for 4 hrs. By that time TLC showed thecompletion of the reaction (ethylacetate:hexane, 60:40). The solvent wasevaporated in vacuum. Residue obtained was placed on a flash column andeluted with ethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

[0155]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0156]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).

[0157]5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0158]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as awhite foam (14.6 g, 80%).

[0159] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0160] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH).This mixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

[0161] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0162] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

[0163]5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0164] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

[0165] 2′-(Aminooxyethoxy) Nucleoside Amidites

[0166] 2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

[0167]N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0168] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2N-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0169] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

[0170] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

[0171] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

[0172] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) isslowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-, 2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

[0173] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine

[0174] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

[0175]5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0176] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2

[0177] Oligonucleotide Synthesis

[0178] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 380B) using standard phosphoramidite chemistrywith oxidation by iodine.

[0179] Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution. Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

[0180] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0181] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0182] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No., 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0183] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0184] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0185] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0186] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3

[0187] Oligonucleoside Synthesis

[0188] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethyl-hydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligo-nucleosides, also identified as amide-4 linked oligonucleo-sides,as well as mixed backbone compounds having, for instance, alternatingMMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0189] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0190] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0191] PNA Synthesis

[0192] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

[0193] Synthesis of Chimeric Oligonucleotides

[0194] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0195] [2′-O-Me]-[2′-deoxyl-[2′-O-Me] Chimeric

[0196] Phosphorothioate Oligonucleotides

[0197] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligo-nucleotide segments are synthesizedusing an Applied Biosystems automated DNA synthesizer Model 380B, asabove. Oligonucleotides are synthesized using the automated synthesizerand 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphor-amidite for the DNAportion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′and 3′ wings. The standard synthesis cycle is modified by increasing thewait step after the delivery of tetrazole and base to 600 s repeatedfour times for RNA and twice for 2′-O-methyl. The fully protectedoligonucleotide is cleaved from the support and the phosphate group isdeprotected in 3:1 ammonia/ethanol at room temperature overnight thenlyophilized to dryness. Treatment in methanolic ammonia for 24 hrs atroom temperature is then done to deprotect all bases and sample wasagain lyophilized to dryness. The pellet is resuspended in 1M TBAF inTHF for 24 hrs at room temperature to deprotect the 2′ positions. Thereaction is then quenched with 1M TEAA and the sample is then reduced to½ volume by rotovac before being desalted on a G25 size exclusioncolumn. The oligo recovered is then analyzed spectrophotometrically foryield and for purity by capillary electrophoresis and by massspectrometry.

[0198] [2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate

[0199] Oligonucleotides

[0200] [2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxy-ethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[0201] [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0202] [2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphos-phorothioate]-[2′-O-(methoxyethyl) phosphodiesterl chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3, H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0203] Other chimeric oligonucleotides, chimeric oligonucleo-sides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Examaple 6

[0204] Oligonucleotide Isolation

[0205] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

[0206] Oligonucleotide Synthesis—96 Well Plate Format

[0207] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a standard 96 well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

[0208] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄0H at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

[0209] Oligonucleotide Analysis—96 Well Plate Format

[0210] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

[0211] Cell Culture and Oligonucleotide Treatment

[0212] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following 4 cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,Ribonuclease protection assays, or RT-PCR.

[0213] T-24 Cells:

[0214] The transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Gibcco/Life Technologies, Gaithersburg, Md.) supplemented with10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Palcon-Primaria#3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0215] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0216] A549 cells:

[0217] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

[0218] NHDF Cells:

[0219] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville Md.). NHDs were routinely maintainedin Fibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

[0220] HEK Cells:

[0221] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville Md.). HEKs were routinely maintainedin Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

[0222] Treatment with Antisense Compounds:

[0223] When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired concentration of oligonucleotide. After 4-7hours of treatment, the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

[0224] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10

[0225] Analysis of Oligonucleotide Inhibition of ARA70 Expression

[0226] Antisense modulation of ARA70 expression can be assayed in avariety of ways known in the art. For example, ARA70 mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. Methods of RNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons,Inc., 1993. Northern blot analysis is routine in the art and is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions. Prior to quantitative PCRanalysis, primer-probe sets specific to the target gene being measuredare evaluated for their ability to be “multiplexed” with a GAPDHamplification reaction. In multiplexing, both the target gene and theinternal standard gene GAPDH are amplified concurrently in a singlesample. In this analysis, mRNA isolated from untreated cells is seriallydiluted. Each dilution is amplified in the presence of primer-probe setsspecific for GAPDH only, target gene only (“single-plexing”), or both(multiplexing). Following PCR amplification, standard curves of GAPDHand target mRNA signal as a function of dilution are generated from boththe single-plexed and multiplexed samples. If both the slope andcorrelation coefficient of the GAPDH and target signals generated fromthe multiplexed samples fall within 10% of their corresponding valuesgenerated from the single-plexed samples, the primer-probe set specificfor that target is deemed as multiplexable. Other methods of PCR arealso known in the art.

[0227] Protein levels of ARA70 can be quantitated in a variety of wayswell known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), ELISA or fluorescence-activated cell sorting(FACS). Antibodies directed to ARA70 can be identified and obtained froma variety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

[0228] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998. Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11

[0229] Poly(A)+ mRNA Isolation

[0230] Poly(A)+ mRNA was isolated according to Miura et al., Clin.Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolationare taught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C. was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

[0231] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12

[0232] Total RNA Isolation

[0233] Total mRNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 100 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 100 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPEwas then added to each well of the RNEASY 96™ plate and the vacuumapplied for a period of 15 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 10 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 60 μL water into each well, incubating1 minute, and then applying the vacuum for 30 seconds. The elution stepwas repeated with an additional 60 μL water.

[0234] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

[0235] Real-time Quantitative PCR Analysis of ARA70 mRNA Levels

[0236] Quantitation of ARA70 mRNA levels was determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Poster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE, FAM, or VIC, obtained from either Operon TechnologiesInc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ 7700 Sequence Detection System. In each assay,a series of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

[0237] PCR reagents were obtained from PE-Applied Biosystems, FosterCity, Calif. RT-PCR reactions were carried out by adding 25 μL PCRcocktail (1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTPand dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer,and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLpoly(A) mRNA solution. The RT reaction was carried out by incubation for30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension).

[0238] Probes and primers to human ARA70 were designed to hybridize to ahuman ARA70 sequence, using published sequence information (GenBankaccession number L49399, incorporated herein as SEQ ID NO: 3). For humanARA70 the PCR primers were: forward primer: TGCGAGAGGTCAAAGCTCAGA (SEQID NO: 4) reverse primer: TGCTGAGCCTGCTGTTGAAGT (SEQ ID NO: 5) and thePCR probe was: FAM-TCACAGTTGCATAAGCCGTCACCTGGA-TAMRA (SEQ ID NO: 6)where FAM (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye. For human GAPDH the PCR primers were:forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7) reverse primer:GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the PCR probe was: 5′JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 9) where JOE (PE-AppliedBiosystems, Foster City, Calif.) is the fluorescent reporter dye) andTAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Example 14

[0239] Northern Blot Analysis of ARA70 mRNA Levels

[0240] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UW cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0241] To detect human ARA70, a human ARA70 specific probe was preparedby PCR using the forward primer TGCGAGAGGTCAAAGCTCAGA (SEQ ID NO: 4) andthe reverse primer TGCTGAGCCTGCTGTTGAAGT (SEQ ID NO: 5) To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)RNA (Clontech, Palo Alto, Calif.).

[0242] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15

[0243] Antisense Inhibition of Human ARA70 Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0244] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humanARA70 RNA, using published sequences (GenBank accession number AA578140,the complement of which is incorporated herein as SEQ ID NO: 10, GenBankaccession number AA090644, incorporated herein as SEQ ID NO: 11, andGenBank accession number X77548, incorporated herein as SEQ ID NO: 12).The oligonucleotides are shown in Table 1. “Target site” indicates thefirst (5′-most) nucleotide number on the particular target sequence towhich the oligonucleotide binds. All compounds in Table 1 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humanARA70 mRNA levels by quantitative real-time PCR as described in otherexamples herein. Data are averages from two experiments. If present,“N.D.” indicates “no data”. TABLE 1 Inhibition of human APA70 mRNAlevels by chimeric phosphorothioate oligonucleotides having 2′-MOE wingsand a deoxy gap TARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITESEQUENCE % INHIB NO 114445 5′ UTR 10 7 tcttactctttaagctgatg 32 13 1144465′ UTR 10 44 ccacactaacctacggccca 33 14 114442 5′ UTR 11 7cccgcccacgcgtcacacgg 73 15 114443 5′ UTR 11 11 tcgtcccgcccacgcgtcac 3616 114444 5′ UTR 11 44 gacccgagtgcgaaaggtcc 36 17 114369 5′ UTR 12 44ccaggtccgagtgcctctcc 44 18 114370 Start 12 69 gaaggtattcattctcctca 86 19Codon 114371 Coding 12 85 tgccactctggtcttggaag 71 20 114372 Coding 12125 gcatcactacacctcaaaag 81 21 114373 Coding 12 189 gttatctttaatttgctgtt45 22 114374 Coding 12 257 tcacggcttctaagacattc 35 23 114375 Coding 12318 ctgttgaagtgtctcctctt 34 24 114376 Coding 12 350 tggcccaataacgagtagag37 25 114377 Coding 12 413 gagacttgattggctagatc 68 26 114378 Coding 12421 ggcacacagagacttgattg 53 27 114379 Coding 12 431 agtctctccaggcacacaga56 28 114380 Coding 12 461 gaatcttcaggcttaagggt 13 29 114381 Coding 12493 taattgtgtcagcttcaaag 21 30 114382 Coding 12 519 aaatgtggtgatggtctggc40 31 114383 Coding 12 554 aagtgctcaggaatttgaat 27 32 114384 Coding 12557 atcaagtgctcaggaatttg 29 33 114385 Coding 12 748 tttgggtaagccagtcctgg69 34 114386 Coding 12 793 aattgcaggctctggaagaa 33 35 114387 Coding 12892 tagtagtggaatggctgtta 39 36 114388 Coding 12 1119ctggtttccctgacagttgg 73 37 114389 Coding 12 1159 acttcagattggccaggttt 1138 114390 Coding 12 1226 agccaatcctctgtaaccat 74 39 114391 Coding 121244 tcctgatggttctggacaag 53 40 114392 Coding 12 1273ctctgcacacctcctctacc 52 41 114393 Coding 12 1366 ttccttctttcttcagaagc 5642 114394 Coding 12 1421 tctttatgcttctcaggctc 67 43 114395 Coding 121504 caattctagaaggagtcatt 28 44 114396 Coding 12 1527ctttatgacttggaaggaat 31 45 114397 Coding 12 1600 cagtaccaggcacttccttg 8746 114398 Coding 12 1624 acttctgtttgccagctctg 64 47 114399 Coding 121702 ggttgcccatcttctttcct 70 48 114400 Coding 12 1751gccttctttcgaagcagcca 47 49 114401 Coding 12 1792 tatgttcctcctgtagaggt 6050 114402 Stop 12 1911 tccattccttcacatctgta 58 51 Codon 114403 3′ UTR 121947 tgtgtgataatcagcagaaa 63 52 114404 3′ UTR 12 1978atttggcaagctgcagtcac 75 53 114405 3′ UTR 12 2022 caggagaagaactaagctaa 6254 114406 3′ UTR 12 2117 aactgaatcacctcagcatg 83 55 114407 3′ UTR 122122 gagggaactgaatcacctca 84 56 114408 3′ UTR 12 2231tacttttagtaacgacccaa 71 57 114409 3′ UTR 12 2236 taatgtacttttagtaacga 4758 114410 3′ UTR 12 2298 aacgttagggaagtttacta 54 59 114411 3′ UTR 122309 tagggcaattaaacgttagg 65 60 114412 3′ UTR 12 2349accactaacaaaagaaaacc 29 61 114413 3′ UTR 12 2464 atgtcaaaaattgccaagat 6162 114414 3′ UTR 12 2510 tgaagaataatgtagaacta 8 63 114415 3′ UTR 12 2621tagaattacctcaaaaatag 34 64 114416 3′ UTR 12 2641 tgtagaatattttaaaaggc 6165 114417 3′ UTR 12 2695 gtcttttgcctatttgctta 88 66 114418 3′ UTR 122701 caacatgtcttttgcctatt 86 67 114419 3′ UTR 12 2724tttcaagtactatactacat 54 68 114420 3′ UTR 12 2725 gtttcaagtactatactaca 7669 114421 3′ UTR 12 2778 aagctcagcttccatttaca 82 70 114422 3′ UTR 122811 tgactttatccctacttaaa 62 71 114423 3′ UTR 12 2822cagtgagaggatgactttat 68 72 114424 3′ UTR 12 2832 gctgtgcttgcagtgagagg 7173 114425 3′ UTR 12 3070 ccggtaagaggcagttggaa 66 74 114426 3′ UTR 123078 aagtatttccggtaagaggc 89 75 114427 3′ UTR 12 3090aagttcccccagaagtattt 16 76 114428 3′ UTR 12 3115 atggcaatgacattttggga 8577 114429 3′ UTR 12 3121 agctgtatggcaatgacatt 77 78 114430 3′ UTR 123127 tggtgaagctgtatggcaat 70 79 114431 3′ UTR 12 3139ttcaaagaactctggtgaag 0 80 114432 3′ UTR 12 3222 ttgcccaccacctttatata 5581 114433 3′ UTR 12 3230 cattgtttttgcccaccacc 74 82 114434 3′ UTR 123241 aaggctccttacattgtttt 84 83 114435 3′ UTR 12 3267cagagctgcaactcaagata 77 84 114436 3′ UTR 12 3276 aagaaactacagagctgcaa 8185 114437 3′ UTR 12 3278 tcaagaaactacagagctgc 80 86 114438 3′ UTR 123297 aaatacagtgtgtttggcct 84 87 114439 3′ UTR 12 3318taagggaaattatattttga 0 88 114440 3′ UTR 12 3319 ttaagggaaattatattttg 089 114441 3′ UTR 12 3322 tgattaagggaaattatatt 0 90

[0245] As shown in Table 1, SEQ ID NOs 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 31, 32, 33, 34, 35, 36, 37, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 77, 78, 79,81, 82, 83, 84, 85, 86 and 87 demonstrated at least 25% inhibition ofhuman ARA70 expression in this assay and are therefore preferred.

Example 16

[0246] Western Blot Analysis of ARA70 Protein Levels

[0247] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to ARA70 is used, with aradiolabelled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 90 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcgctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2atgcattctg cccccaagga 20 3 1845 DNA Homo sapiens CDS (1)...(1845) 3 atgaat acc ttc caa gac cag agt ggc agc tcc agt aat aga gaa ccc 48 Met AsnThr Phe Gln Asp Gln Ser Gly Ser Ser Ser Asn Arg Glu Pro 1 5 10 15 cttttg agg tgt agt gat gca cgg agg gac ttg gag ctt gct att ggt 96 Leu LeuArg Cys Ser Asp Ala Arg Arg Asp Leu Glu Leu Ala Ile Gly 20 25 30 gga gttctc cgg gct gaa cag caa att aaa gat aac ttg cga gag gtc 144 Gly Val LeuArg Ala Glu Gln Gln Ile Lys Asp Asn Leu Arg Glu Val 35 40 45 aaa gct cagatt cac agt tgc ata agc cgt cac ctg gaa tgt ctt aga 192 Lys Ala Gln IleHis Ser Cys Ile Ser Arg His Leu Glu Cys Leu Arg 50 55 60 agc cgt gag gtatgg ctg tat gaa cag gtg gac ctt att tat cag ctt 240 Ser Arg Glu Val TrpLeu Tyr Glu Gln Val Asp Leu Ile Tyr Gln Leu 65 70 75 80 aaa gag gag acactt caa cag cag gct cag cag ctc tac tcg tta ttg 288 Lys Glu Glu Thr LeuGln Gln Gln Ala Gln Gln Leu Tyr Ser Leu Leu 85 90 95 ggc cag ttc aat tgtctt act cat caa ctg gag tgt acc caa aac aaa 336 Gly Gln Phe Asn Cys LeuThr His Gln Leu Glu Cys Thr Gln Asn Lys 100 105 110 gat cta gcc aat caagtc tct gtg tgc ctg gag aga ctg ggc agt ttg 384 Asp Leu Ala Asn Gln ValSer Val Cys Leu Glu Arg Leu Gly Ser Leu 115 120 125 acc ctt aag cct gaagat tca act gtc ctg ctc ttt gaa gct gac aca 432 Thr Leu Lys Pro Glu AspSer Thr Val Leu Leu Phe Glu Ala Asp Thr 130 135 140 att act ctg cgc cagacc atc acc aca ttt ggg tct ctc aaa acc att 480 Ile Thr Leu Arg Gln ThrIle Thr Thr Phe Gly Ser Leu Lys Thr Ile 145 150 155 160 caa att cct gagcac ttg atg gct cat gct agt tca gca aat att ggg 528 Gln Ile Pro Glu HisLeu Met Ala His Ala Ser Ser Ala Asn Ile Gly 165 170 175 ccc ttc ctg gagaag aga ggc tgt atc tcc atg cca gag cag aag tca 576 Pro Phe Leu Glu LysArg Gly Cys Ile Ser Met Pro Glu Gln Lys Ser 180 185 190 gca tcc ggt attgta gct gtc cct ttc agc gaa tgg ctc ctt gga agc 624 Ala Ser Gly Ile ValAla Val Pro Phe Ser Glu Trp Leu Leu Gly Ser 195 200 205 aaa cct gcc agtggt tat caa gct cct tac ata ccc agc acc gac ccc 672 Lys Pro Ala Ser GlyTyr Gln Ala Pro Tyr Ile Pro Ser Thr Asp Pro 210 215 220 cag gac tgg cttacc caa aag cag acc ttg gag aac agt cag act tct 720 Gln Asp Trp Leu ThrGln Lys Gln Thr Leu Glu Asn Ser Gln Thr Ser 225 230 235 240 tcc aga gcctgc aat ttc ttc aat aat gtc ggg gga aac cta aag ggc 768 Ser Arg Ala CysAsn Phe Phe Asn Asn Val Gly Gly Asn Leu Lys Gly 245 250 255 tta gaa aactgg ctc ctc aag agt gaa aaa tca agt tat caa aag tgt 816 Leu Glu Asn TrpLeu Leu Lys Ser Glu Lys Ser Ser Tyr Gln Lys Cys 260 265 270 aac agc cattcc act act agt tct ttc tcc att gaa atg gaa aag gtt 864 Asn Ser His SerThr Thr Ser Ser Phe Ser Ile Glu Met Glu Lys Val 275 280 285 gga gat caagag ctt cct gat caa gat gag atg gac cta tca gat tgg 912 Gly Asp Gln GluLeu Pro Asp Gln Asp Glu Met Asp Leu Ser Asp Trp 290 295 300 cta gtg actccc cag gaa tcc cat aag ctg cgg aag cct gag aat ggc 960 Leu Val Thr ProGln Glu Ser His Lys Leu Arg Lys Pro Glu Asn Gly 305 310 315 320 agt cgtgaa acc agt gag aag ttt aag ctc tta ttc cag tcc tat aat 1008 Ser Arg GluThr Ser Glu Lys Phe Lys Leu Leu Phe Gln Ser Tyr Asn 325 330 335 gtg aatgat tgg ctt gtc aag act gac tcc tgt acc aac tgt cag gga 1056 Val Asn AspTrp Leu Val Lys Thr Asp Ser Cys Thr Asn Cys Gln Gly 340 345 350 aac cagccc aaa ggt gtg gag att gaa aac ctg ggc aat ctg aag tgc 1104 Asn Gln ProLys Gly Val Glu Ile Glu Asn Leu Gly Asn Leu Lys Cys 355 360 365 ctg aatgac cac ttg gag gcc aag aaa cca ttg tcc acc ccc agc atg 1152 Leu Asn AspHis Leu Glu Ala Lys Lys Pro Leu Ser Thr Pro Ser Met 370 375 380 gtt acagag gat tgg ctt gtc cag aac cat cag gac cca tgt aag gta 1200 Val Thr GluAsp Trp Leu Val Gln Asn His Gln Asp Pro Cys Lys Val 385 390 395 400 gaggag gtg tgc aga gcc aat gag ccc tgc aca agc ttt gca gag tgt 1248 Glu GluVal Cys Arg Ala Asn Glu Pro Cys Thr Ser Phe Ala Glu Cys 405 410 415 gtgtgt gat gag aat tgt gag aag gag gct ctg tat aag tgg ctt ctg 1296 Val CysAsp Glu Asn Cys Glu Lys Glu Ala Leu Tyr Lys Trp Leu Leu 420 425 430 aagaaa gaa gga aag gat aaa aat ggg atg cct gtg gaa ccc aaa cct 1344 Lys LysGlu Gly Lys Asp Lys Asn Gly Met Pro Val Glu Pro Lys Pro 435 440 445 gagcct gag aag cat aaa gat tcc ctg aat atg tgg ctc tgt cct aga 1392 Glu ProGlu Lys His Lys Asp Ser Leu Asn Met Trp Leu Cys Pro Arg 450 455 460 aaagaa gta ata gaa caa act aaa gca cca aag gca atg act cct tct 1440 Lys GluVal Ile Glu Gln Thr Lys Ala Pro Lys Ala Met Thr Pro Ser 465 470 475 480aga att gct gat tcc ttc caa gtc ata aag aac agc ccc ttg tcg gag 1488 ArgIle Ala Asp Ser Phe Gln Val Ile Lys Asn Ser Pro Leu Ser Glu 485 490 495tgg ctt atc agg ccc cca tac aaa gaa gga agt ccc aag gaa gtg cct 1536 TrpLeu Ile Arg Pro Pro Tyr Lys Glu Gly Ser Pro Lys Glu Val Pro 500 505 510ggt act gaa gac aga gct ggc aaa cag aag ttt aaa agc ccc atg aat 1584 GlyThr Glu Asp Arg Ala Gly Lys Gln Lys Phe Lys Ser Pro Met Asn 515 520 525act tcc tgg tgt tcc ttt aac aca gct gac tgg gtc ctg cca gga aag 1632 ThrSer Trp Cys Ser Phe Asn Thr Ala Asp Trp Val Leu Pro Gly Lys 530 535 540aag atg ggc aac ctc agc cag tta tct tct gga gaa gac aag tgg ctg 1680 LysMet Gly Asn Leu Ser Gln Leu Ser Ser Gly Glu Asp Lys Trp Leu 545 550 555560 ctt cga aag aag gcc cag gaa gta tta ctt aat tca cct cta cag gag 1728Leu Arg Lys Lys Ala Gln Glu Val Leu Leu Asn Ser Pro Leu Gln Glu 565 570575 gaa cat aac ttc ccc cca gac cat tat ggc ctc cct gca gtt tgt gat 1776Glu His Asn Phe Pro Pro Asp His Tyr Gly Leu Pro Ala Val Cys Asp 580 585590 ctc ttt gcc tgt atg cag ctt aaa gtt gat aaa gag aag tgg tta tat 1824Leu Phe Ala Cys Met Gln Leu Lys Val Asp Lys Glu Lys Trp Leu Tyr 595 600605 cga act cct cta cag atg tga 1845 Arg Thr Pro Leu Gln Met 610 4 21DNA Artificial Sequence PCR Primer 4 tgcgagaggt caaagctcag a 21 5 21 DNAArtificial Sequence PCR Primer 5 tgctgagcct gctgttgaag t 21 6 27 DNAArtificial Sequence PCR Probe 6 tcacagttgc ataagccgtc acctgga 27 7 19DNA Artificial Sequence PCR Primer 7 gaaggtgaag gtcggagtc 19 8 20 DNAArtificial Sequence PCR Primer 8 gaagatggtg atgggatttc 20 9 20 DNAArtificial Sequence PCR Probe 9 caagcttccc gttctcagcc 20 10 381 DNA Homosapiens CDS (137)...(381) 10 cgtcgacatc agcttaaaga gtaagacaat tgaactctgcctttgggccg taggttagtg 60 tggggccgtg tctcagtcca cccaaggtct cctcggatcgcctggagagg cactcggacc 120 tggagcagtg aggaga atg aat acc ttc caa gac cagagt ggc agc tcc agt 172 Met Asn Thr Phe Gln Asp Gln Ser Gly Ser Ser Ser1 5 10 aat aga gaa ccc ctt ttg agg tgt agt gat gca cgg agg gac ttg gag220 Asn Arg Glu Pro Leu Leu Arg Cys Ser Asp Ala Arg Arg Asp Leu Glu 1520 25 ctt gct att ggt gga gtt ctc cgg gct gaa cag caa att aaa gat aac268 Leu Ala Ile Gly Gly Val Leu Arg Ala Glu Gln Gln Ile Lys Asp Asn 3035 40 ttg cga gag gtc aaa gct cag att cac agt tgc ata agc cgt cac ctg316 Leu Arg Glu Val Lys Ala Gln Ile His Ser Cys Ile Ser Arg His Leu 4550 55 60 gaa tgt ctt aga agc cgt gag gta tgg tat gta tga aca ggt gga cct364 Glu Cys Leu Arg Ser Arg Glu Val Trp Tyr Val Thr Gly Gly Pro 65 70 75tat tta tca ggt cga gc 381 Tyr Leu Ser Gly Arg 80 11 221 DNA Homosapiens CDS (138)...(221) 11 gagttgccgt gtgacgcgtg ggcgggacga ggcccgggctcggggacctt tcgcactcgg 60 gtcaggggta aagcagcctt tcgcttgccg ggcagctggtgagtcggtga ccttggcctg 120 ttaggagcag ttaggag atg aat acc ttc caa gac cagagt ggc agc tcc 170 Met Asn Thr Phe Gln Asp Gln Ser Gly Ser Ser 1 5 10agt aat aga gaa ccc ctt ttg agg ttt agt gat gca cgg agg gac ttg 218 SerAsn Arg Glu Pro Leu Leu Arg Phe Ser Asp Ala Arg Arg Asp Leu 15 20 25 agc221 Ser 12 3436 DNA Homo sapiens CDS (77)...(1921) 12 caatcgcgaccctcagtcca cccaaggtct cctcggatcg cctggagagg cactcggacc 60 tggagcagtgaggaga atg aat acc ttc caa gac cag agt ggc agc tcc agt 112 Met Asn ThrPhe Gln Asp Gln Ser Gly Ser Ser Ser 1 5 10 aat aga gaa ccc ctt ttg aggtgt agt gat gca cgg agg gac ttg gag 160 Asn Arg Glu Pro Leu Leu Arg CysSer Asp Ala Arg Arg Asp Leu Glu 15 20 25 ctt gct att ggt gga gtt ctc cgggct gaa cag caa att aaa gat aac 208 Leu Ala Ile Gly Gly Val Leu Arg AlaGlu Gln Gln Ile Lys Asp Asn 30 35 40 ttg cga gag gtc aaa gct cag att cacagt tgc ata agc cgt cac ctg 256 Leu Arg Glu Val Lys Ala Gln Ile His SerCys Ile Ser Arg His Leu 45 50 55 60 gaa tgt ctt aga agc cgt gag gta tggctg tat gaa cag gtg gac ctt 304 Glu Cys Leu Arg Ser Arg Glu Val Trp LeuTyr Glu Gln Val Asp Leu 65 70 75 att tat cag ctt aaa gag gag aca ctt caacag cag gct cag cag ctc 352 Ile Tyr Gln Leu Lys Glu Glu Thr Leu Gln GlnGln Ala Gln Gln Leu 80 85 90 tac tcg tta ttg ggc cag ttc aat tgt ctt actcat caa ctg gag tgt 400 Tyr Ser Leu Leu Gly Gln Phe Asn Cys Leu Thr HisGln Leu Glu Cys 95 100 105 acc caa aac aaa gat cta gcc aat caa gtc tctgtg tgc ctg gag aga 448 Thr Gln Asn Lys Asp Leu Ala Asn Gln Val Ser ValCys Leu Glu Arg 110 115 120 ctg ggc agt ttg acc ctt aag cct gaa gat tcaact gtc ctg ctc ttt 496 Leu Gly Ser Leu Thr Leu Lys Pro Glu Asp Ser ThrVal Leu Leu Phe 125 130 135 140 gaa gct gac aca att act ctg cgc cag accatc acc aca ttt ggg tct 544 Glu Ala Asp Thr Ile Thr Leu Arg Gln Thr IleThr Thr Phe Gly Ser 145 150 155 ctc aaa acc att caa att cct gag cac ttgatg gct cat gct agt tca 592 Leu Lys Thr Ile Gln Ile Pro Glu His Leu MetAla His Ala Ser Ser 160 165 170 gca aat att ggg ccc ttc ctg gag aag agaggc tgt atc tcc atg cca 640 Ala Asn Ile Gly Pro Phe Leu Glu Lys Arg GlyCys Ile Ser Met Pro 175 180 185 gag cag aag tca gca tcc ggt att gta gctgtc cct ttc agc gaa tgg 688 Glu Gln Lys Ser Ala Ser Gly Ile Val Ala ValPro Phe Ser Glu Trp 190 195 200 ctc ctt gga agc aaa cct gcc agt ggt tatcaa gct cct tac ata ccc 736 Leu Leu Gly Ser Lys Pro Ala Ser Gly Tyr GlnAla Pro Tyr Ile Pro 205 210 215 220 agc acc gac ccc cag gac tgg ctt acccaa aag cag acc ttg gag aac 784 Ser Thr Asp Pro Gln Asp Trp Leu Thr GlnLys Gln Thr Leu Glu Asn 225 230 235 agt cag act tct tcc aga gcc tgc aatttc ttc aat aat gtc ggg gga 832 Ser Gln Thr Ser Ser Arg Ala Cys Asn PhePhe Asn Asn Val Gly Gly 240 245 250 aac cta aag ggc tta gaa aac tgg ctcctc aag agt gaa aaa tca agt 880 Asn Leu Lys Gly Leu Glu Asn Trp Leu LeuLys Ser Glu Lys Ser Ser 255 260 265 tat caa aag tgt aac agc cat tcc actact agt tct ttc tcc att gaa 928 Tyr Gln Lys Cys Asn Ser His Ser Thr ThrSer Ser Phe Ser Ile Glu 270 275 280 atg gaa aag gtt gga gat caa gag cttcct gat caa gat gag atg gac 976 Met Glu Lys Val Gly Asp Gln Glu Leu ProAsp Gln Asp Glu Met Asp 285 290 295 300 cta tca gat tgg cta gtg act ccccag gaa tcc cat aag ctg cgg aac 1024 Leu Ser Asp Trp Leu Val Thr Pro GlnGlu Ser His Lys Leu Arg Asn 305 310 315 gct gag aat ggc agt cgt gaa accagt gag aag ttt aag ctc tta ttc 1072 Ala Glu Asn Gly Ser Arg Glu Thr SerGlu Lys Phe Lys Leu Leu Phe 320 325 330 cag tcc tat aat gtg aat gat tggctt gtc aag act gac tcc tgt acc 1120 Gln Ser Tyr Asn Val Asn Asp Trp LeuVal Lys Thr Asp Ser Cys Thr 335 340 345 aac tgt cag gga aac cag ccc aaaggt gtg gag att gaa aac ctg gcc 1168 Asn Cys Gln Gly Asn Gln Pro Lys GlyVal Glu Ile Glu Asn Leu Ala 350 355 360 aat ctg aag tgc ctg aat gac cacttg gag gcc aag aaa cca ttg tcc 1216 Asn Leu Lys Cys Leu Asn Asp His LeuGlu Ala Lys Lys Pro Leu Ser 365 370 375 380 acc ccc agc atg gtt aca gaggat tgg ctt gtc cag aac cat cag gac 1264 Thr Pro Ser Met Val Thr Glu AspTrp Leu Val Gln Asn His Gln Asp 385 390 395 cca tgt aag gta gag gag gtgtgc aga gcc aat gag ccc tgc aca agc 1312 Pro Cys Lys Val Glu Glu Val CysArg Ala Asn Glu Pro Cys Thr Ser 400 405 410 ttt gca gag tgt gtg tgt gatgag aat tgt gag aag gag gct ctg tat 1360 Phe Ala Glu Cys Val Cys Asp GluAsn Cys Glu Lys Glu Ala Leu Tyr 415 420 425 aag tgg ctt ctg aag aaa gaagga aag gat aaa aat ggg atg cct gtg 1408 Lys Trp Leu Leu Lys Lys Glu GlyLys Asp Lys Asn Gly Met Pro Val 430 435 440 gaa ccc aaa cct gag cct gagaag cat aaa gat tcc ctg aat atg tgg 1456 Glu Pro Lys Pro Glu Pro Glu LysHis Lys Asp Ser Leu Asn Met Trp 445 450 455 460 ctc tgt cct aga aaa gaagta ata gaa caa act aaa gca cca aag gca 1504 Leu Cys Pro Arg Lys Glu ValIle Glu Gln Thr Lys Ala Pro Lys Ala 465 470 475 atg act cct tct aga attgct gat tcc ttc caa gtc ata aag aac agc 1552 Met Thr Pro Ser Arg Ile AlaAsp Ser Phe Gln Val Ile Lys Asn Ser 480 485 490 ccc ttg tcg gag tgg cttatc agg ccc cca tac aaa gaa gga agt ccc 1600 Pro Leu Ser Glu Trp Leu IleArg Pro Pro Tyr Lys Glu Gly Ser Pro 495 500 505 aag gaa gtg cct ggt actgaa gac aga gct ggc aaa cag aag ttt aaa 1648 Lys Glu Val Pro Gly Thr GluAsp Arg Ala Gly Lys Gln Lys Phe Lys 510 515 520 agc ccc atg aat act tcctgg tgt tcc ttt aac aca gct gac tgg gtc 1696 Ser Pro Met Asn Thr Ser TrpCys Ser Phe Asn Thr Ala Asp Trp Val 525 530 535 540 ctg cca gga aag aagatg ggc aac ctc agc cag tta tct tct gga gaa 1744 Leu Pro Gly Lys Lys MetGly Asn Leu Ser Gln Leu Ser Ser Gly Glu 545 550 555 gac aag tgg ctg cttcga aag aag gcc cag gaa gta tta ctt aat tca 1792 Asp Lys Trp Leu Leu ArgLys Lys Ala Gln Glu Val Leu Leu Asn Ser 560 565 570 cct cta cag gag gaacat aac ttc ccc cca gac cat tat ggc ctc cct 1840 Pro Leu Gln Glu Glu HisAsn Phe Pro Pro Asp His Tyr Gly Leu Pro 575 580 585 gca gtt tgt gat ctcttt gcc tgt atg cag ctt aaa gtt gat aaa gag 1888 Ala Val Cys Asp Leu PheAla Cys Met Gln Leu Lys Val Asp Lys Glu 590 595 600 aag tgg tta tat cgaact cct cta cag atg tga aggaatggac aagagttgag 1941 Lys Trp Leu Tyr ArgThr Pro Leu Gln Met 605 610 cagcctttct gctgattatc acacatcatg agctgagtgactgcagcttg ccaaatcttt 2001 gtgtttctgg gtctgaccaa ttagcttagt tcttctcctgcctaattttg aactagtaaa 2061 gcaaagtgag tcatcagatt atgagttact gtttaaaagaaaaatgctgt ttattcatgc 2121 tgaggtgatt cagttccctc cttcttacag aagtattttaattcacccca cactagaaat 2181 gcagcatctt tgtggacgtc tttttcacaa gcctccaaggctccttagat tgggtcgtta 2241 ctaaaagtac attaaaacac tcttgtttat cgaagtatattgatgtattc taaagctagt 2301 aaacttccct aacgtttaat tgccctacag atgcttctcttgctgtgggt tttcttttgt 2361 tagtggtctg aaataattat tttcctgttc tattaatacatagtgtattt tgcacaaaaa 2421 aattaacctg gtcaatagtg attaccaaaa tatatattaataatcttggc aatttttgac 2481 attaattatg aaacatttta gcccacgtta gttctacattattcttcact taaactcagc 2541 tactgcaaat tttgtctttc tgtaaatgtt attaaaatatccagtgagct ctttagaagg 2601 actcagtatt atttcaagac tatttttgag gtaattctagccttttaaaa tattctacag 2661 acctacgggg cttaaaagaa ccccagtacc gactaagcaaataggcaaaa gacatgttgg 2721 aaatgtagta tagtacttga aacagtcact atcatagggataattggtgc atcctgtgta 2781 aatggaagct gagcttgaca cctggtgctt ttaagtagggataaagtcat cctctcactg 2841 caagcacagc atacctgtac ctccaaaagt gacgttttagtgaacaggcc gttttcaaca 2901 cttgtgcctt ggggtgttca ttgaagcttt gtgaaaactactgatgtttt ctcagtctcc 2961 ttaaagttac gtccatgctt taaaatgtct gtgtaggagagaagtggggt ttataatgtt 3021 ttctctaaga tatctttgct gctttccaga ctttgaaactattaagcttt ccaactgcct 3081 cttaccggaa atacttctgg gggaacttca tggtcccaaaatgtcattgc catacagctt 3141 caccagagtt ctttgaacca cagctgaaaa gagctttgtattatttttta attccctccc 3201 cagatatcat ttaggagtat tatataaagg tggtgggcaaaaacaatgta aggagccttt 3261 ccagttatct tgagttgcag ctctgtagtt tcttgaggccaaacacactg tatttgtcaa 3321 aatataattt cccttaatca ctatgttaat gagtatgtaaaacattcttt tgcattgatg 3381 aattttgtat ctgcttccct taaagcataa cagccataaaaaaaaaaaaa aaaaa 3436 13 20 DNA Artificial Sequence AntisenseOligonucleotide 13 tcttactctt taagctgatg 20 14 20 DNA ArtificialSequence Antisense Oligonucleotide 14 ccacactaac ctacggccca 20 15 20 DNAArtificial Sequence Antisense Oligonucleotide 15 cccgcccacg cgtcacacgg20 16 20 DNA Artificial Sequence Antisense Oligonucleotide 16 tcgtcccgcccacgcgtcac 20 17 20 DNA Artificial Sequence Antisense Oligonucleotide 17gacccgagtg cgaaaggtcc 20 18 20 DNA Artificial Sequence AntisenseOligonucleotide 18 ccaggtccga gtgcctctcc 20 19 20 DNA ArtificialSequence Antisense Oligonucleotide 19 gaaggtattc attctcctca 20 20 20 DNAArtificial Sequence Antisense Oligonucleotide 20 tgccactctg gtcttggaag20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21 gcatcactacacctcaaaag 20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22gttatcttta atttgctgtt 20 23 20 DNA Artificial Sequence AntisenseOligonucleotide 23 tcacggcttc taagacattc 20 24 20 DNA ArtificialSequence Antisense Oligonucleotide 24 ctgttgaagt gtctcctctt 20 25 20 DNAArtificial Sequence Antisense Oligonucleotide 25 tggcccaata acgagtagag20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26 gagacttgattggctagatc 20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27ggcacacaga gacttgattg 20 28 20 DNA Artificial Sequence AntisenseOligonucleotide 28 agtctctcca ggcacacaga 20 29 20 DNA ArtificialSequence Antisense Oligonucleotide 29 gaatcttcag gcttaagggt 20 30 20 DNAArtificial Sequence Antisense Oligonucleotide 30 taattgtgtc agcttcaaag20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31 aaatgtggtgatggtctggc 20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32aagtgctcag gaatttgaat 20 33 20 DNA Artificial Sequence AntisenseOligonucleotide 33 atcaagtgct caggaatttg 20 34 20 DNA ArtificialSequence Antisense Oligonucleotide 34 tttgggtaag ccagtcctgg 20 35 20 DNAArtificial Sequence Antisense Oligonucleotide 35 aattgcaggc tctggaagaa20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36 tagtagtggaatggctgtta 20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37ctggtttccc tgacagttgg 20 38 20 DNA Artificial Sequence AntisenseOligonucleotide 38 acttcagatt ggccaggttt 20 39 20 DNA ArtificialSequence Antisense Oligonucleotide 39 agccaatcct ctgtaaccat 20 40 20 DNAArtificial Sequence Antisense Oligonucleotide 40 tcctgatggt tctggacaag20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41 ctctgcacacctcctctacc 20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42ttccttcttt cttcagaagc 20 43 20 DNA Artificial Sequence AntisenseOligonucleotide 43 tctttatgct tctcaggctc 20 44 20 DNA ArtificialSequence Antisense Oligonucleotide 44 caattctaga aggagtcatt 20 45 20 DNAArtificial Sequence Antisense Oligonucleotide 45 ctttatgact tggaaggaat20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46 cagtaccaggcacttccttg 20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47acttctgttt gccagctctg 20 48 20 DNA Artificial Sequence AntisenseOligonucleotide 48 ggttgcccat cttctttcct 20 49 20 DNA ArtificialSequence Antisense Oligonucleotide 49 gccttctttc gaagcagcca 20 50 20 DNAArtificial Sequence Antisense Oligonucleotide 50 tatgttcctc ctgtagaggt20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51 tccattccttcacatctgta 20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52tgtgtgataa tcagcagaaa 20 53 20 DNA Artificial Sequence AntisenseOligonucleotide 53 atttggcaag ctgcagtcac 20 54 20 DNA ArtificialSequence Antisense Oligonucleotide 54 caggagaaga actaagctaa 20 55 20 DNAArtificial Sequence Antisense Oligonucleotide 55 aactgaatca cctcagcatg20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 gagggaactgaatcacctca 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57tacttttagt aacgacccaa 20 58 20 DNA Artificial Sequence AntisenseOligonucleotide 58 taatgtactt ttagtaacga 20 59 20 DNA ArtificialSequence Antisense Oligonucleotide 59 aacgttaggg aagtttacta 20 60 20 DNAArtificial Sequence Antisense Oligonucleotide 60 tagggcaatt aaacgttagg20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 accactaacaaaagaaaacc 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62atgtcaaaaa ttgccaagat 20 63 20 DNA Artificial Sequence AntisenseOligonucleotide 63 tgaagaataa tgtagaacta 20 64 20 DNA ArtificialSequence Antisense Oligonucleotide 64 tagaattacc tcaaaaatag 20 65 20 DNAArtificial Sequence Antisense Oligonucleotide 65 tgtagaatat tttaaaaggc20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 gtcttttgcctatttgctta 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67caacatgtct tttgcctatt 20 68 20 DNA Artificial Sequence AntisenseOligonucleotide 68 tttcaagtac tatactacat 20 69 20 DNA ArtificialSequence Antisense Oligonucleotide 69 gtttcaagta ctatactaca 20 70 20 DNAArtificial Sequence Antisense Oligonucleotide 70 aagctcagct tccatttaca20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 tgactttatccctacttaaa 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72cagtgagagg atgactttat 20 73 20 DNA Artificial Sequence AntisenseOligonucleotide 73 gctgtgcttg cagtgagagg 20 74 20 DNA ArtificialSequence Antisense Oligonucleotide 74 ccggtaagag gcagttggaa 20 75 20 DNAArtificial Sequence Antisense Oligonucleotide 75 aagtatttcc ggtaagaggc20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 aagttcccccagaagtattt 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77atggcaatga cattttggga 20 78 20 DNA Artificial Sequence AntisenseOligonucleotide 78 agctgtatgg caatgacatt 20 79 20 DNA ArtificialSequence Antisense Oligonucleotide 79 tggtgaagct gtatggcaat 20 80 20 DNAArtificial Sequence Antisense Oligonucleotide 80 ttcaaagaac tctggtgaag20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 ttgcccaccacctttatata 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82cattgttttt gcccaccacc 20 83 20 DNA Artificial Sequence AntisenseOligonucleotide 83 aaggctcctt acattgtttt 20 84 20 DNA ArtificialSequence Antisense Oligonucleotide 84 cagagctgca actcaagata 20 85 20 DNAArtificial Sequence Antisense Oligonucleotide 85 aagaaactac agagctgcaa20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86 tcaagaaactacagagctgc 20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87aaatacagtg tgtttggcct 20 88 20 DNA Artificial Sequence AntisenseOligonucleotide 88 taagggaaat tatattttga 20 89 20 DNA ArtificialSequence Antisense Oligonucleotide 89 ttaagggaaa ttatattttg 20 90 20 DNAArtificial Sequence Antisense Oligonucleotide 90 tgattaaggg aaattatatt20

What is claimed is:
 1. An antisense compound 8 to 30 nucleobases inlength targeted to a nucleic acid molecule encoding ARA70, wherein saidantisense compound specifically hybridizes with and inhibits theexpression of ARA70.
 2. The antisense compound of claim 1 which is anantisense oligonucleotide.
 3. The antisense compound of claim 2 whereinthe antisense oligonucleotide has a sequence comprising SEQ ID NO: 13,14, 15, 16., 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 31, 32, 33,34, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 77, 78, 79, 81, 82, 83, 84, 85, 86 or
 87. 4. Theantisense compound of claim 2 wherein the antisense oligonucleotidecomprises at least one modified internucleoside linkage.
 5. Theantisense compound of claim 4 wherein the modified internucleosidelinkage is a phosphorothioate linkage.
 6. The antisense compound ofclaim 2 wherein the antisense oligonucleotide comprises at least onemodified sugar moiety.
 7. The antisense compound of claim 6 wherein themodified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
 8. Theantisense compound of claim 2 wherein the antisense oligonucleotidecomprises at least one modified nucleobase.
 9. The antisense compound ofclaim 8 wherein the modified nucleobase is a 5-methylcytosine.
 10. Theantisense compound of claim 2 wherein the antisense oligonucleotide is achimeric oligonucleotide.
 11. A composition comprising the antisensecompound of claim 1 and a pharmaceutically acceptable carrier ordiluent.
 12. The composition of claim 11 further comprising a colloidaldispersion system.
 13. The composition of claim 11 wherein the antisensecompound is an antisense oligonucleotide.
 14. A method of inhibiting theexpression of ARA70 in cells or tissues comprising contacting said cellsor tissues with the antisense compound of claim 1 so that expression ofARA70 is inhibited.
 15. A method of treating a human having a disease orcondition associated with ARA70 comprising administering to said animala therapeutically or prophylactically effective amount of the antisensecompound of claim 1 so that expression of ARA70 is inhibited.
 16. Themethod of claim 15 wherein the disease or condition is anendocrinological disorder.
 17. The method of claim 15 wherein thedisease or condition is a prostatic disorder.
 18. The method of claim 15wherein the disease or condition is a hyperproliferative disorder. 19.The method of claim 18 wherein the hyperproliferative disorder iscancer.